Identification and molecular characterization of rice promoters conferring microspore-preferred expression

Thesis for the Degree of Ph.D

Identification and molecular characterization of

rice promoters conferring microspore-preferred

expression

School of Applied Biosciences, Major in Agronomy

The Graduate School

Nguyen, Tien-Dung

December 2015

The Graduate School

Kyungpook National University

Identification and molecular characterization

of rice promoters conferring microspore￾preferred expression

Nguyen, Tien-Dung

School of Applied Biosciences, Major in Agronomy

The Graduate School

Supervised by Professor Lee, Jeong-Dong

Approved as a qualified thesis of Nguyen, Tien-Dung

for the degree of Ph.D

by the Evaluation Committee

December 2015

Chairman __________________

__________________

__________________

__________________

__________________

__________________

The Graduate School Council, Kyungpook National University

Prof. Song, Jong-Tae Prof. Park, Soon-Ki Prof. Jung, Ki-Hong Prof. Park, Dong-Soo Prof. Lee, Jeong-Dong

I

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................II

LIST OF FIGURES .................................................................................................III

LIST OF APPENDIX..........................................................................................................................V

ACKNOWLEDGEMENT.......................................................................................VI

CHAPTER 1. GENERAL INTRODUCTION ......................................................1

CHAPTER 2. IDENTIFICATION AND CHARACTERIZATION OF

MICROSPORE-PREFERRED GENES................................................................19

INTRODUCTION .....................................................................................................19

MATERIALS AND METHODS...............................................................................20

RESULTS ..................................................................................................................27

DISCUSSION............................................................................................................56

CHAPTER 3. FUNCTIONAL VERIFICATION OF MICROSPORE￾PREFERRED PROMOTERS ACTIVITY IN MICROSPORE .........................67

INTRODUCTION .....................................................................................................67

MATERIALS AND METHODS...............................................................................68

RESULTS ..................................................................................................................71

DISCUSSION............................................................................................................85

CHAPTER 4. GENERAL DISCUSSION..............................................................87

REFERRENCES......................................................................................................93

II

LIST OF TABLES

Table 1. Candidate genes exhibiting microspore-preferred expression ....................30

Table 2. The most abundant CREs in RMP promoter region....................................34

Table 3. Organ specific-CREs in RMP promoter region ..........................................35

Table 4. Unique CREs in RMP promoter region ......................................................36

III

LIST OF FIGURES

Figure 1. Diagram of rice (Oryza sativa L.) floral structure..................................... 3

Figure 2. Schematic diagram of the male gametophyte development in

Arabidospsis............................................................................................................... 8

Figure 3. Female gametophyte development in Arabidospsis ................................. 9

Figure 4. Heat map analysis of expression patterns for rice microspore-preferred (RMP)

genes ..................................................................................................................................... 28

Figure 5. Schematic diagram of destination vectors used for plant transformation .31

Figure 6. Frequency of the most abundant CREs in RMP promoter region .............39

Figure 7. Frequency of organ specific-CREs in RMP promoter region.............................. 40

Figure 8. Frequency of unique CREs in RMP promoter region ..................................41

Figure 9. Gus expression driven by RMP promoters during pollen development

in rice .........................................................................................................................46

Figure 10. Confirmation T-DNA insertion in T2 rice transgenic plants...................47

Figure 11. Gus expression driven by the RMP promoters in vegetative organs in

rice..............................................................................................................................48

Figure 12. GUS expression driven by the RMP promoters during pollen

developmental stages in Arabidopsis.........................................................................53

Figure 13. GUS expression driven by the RMP promoters in Arabidospsis at

seedling stage ............................................................................................................. 54

Figure 14. The schematic diagram of prOsLSP10/RMP-SCP:dHA vectors used

for genetic complementation analysis.......................................................................73

Figure 15. The percentage of aberrant pollen grains from non-transformed scp-2

homozygotes (control) and transformed scp-2 hm harboring the proOsLPS10-

SCP:dHA.................................................................................................................... 74

Figure 16. Complementation analysis of scp-2 homozygotes .................................. 75

IV

Figure 17. The percentage of aberrant pollen grains from non-transformed scp-2

homozygotes (control) and transformed scp-2 hm harboring the proRMP1-

SCP:dHA.................................................................................................................... 79

Figure 18. The percentage of aberrant pollen grains from non-transformed scp-2

homozygotes (control) and transformed scp-2 hm harboring the proRMP2-

SCP:dHA.................................................................................................................... 80

Figure 19. The percentage of aberrant pollen grains from non-transformed scp-2

homozygotes (control) and transformed scp-2 hm harboring the proRMP3-

SCP:dHA.................................................................................................................... 81

Figure 20. The percentage of aberrant pollen grains from non-transformed scp-2

homozygotes (control) and transformed scp-2 hm harboring the proRMP6-

SCP:dHA ................................................................................................................... 82

Figure 21. Silique production in complementing lines (transformed scp-2 hm)

compared with scp-2 hm mutant background (control) and wild type plants............ 83

Figure 22. Confirmation of T-DNA insertion in the proRMP-SCP:dHA lines........ 84

V

LIST OF APPENDIX

Appendix 1. Primers used in this study.....................................................................120

Appendix 2. Medium composition used for rice transformation..............................122

Appendix 3. Nucleotide sequences of the OsLPS10 gene. .......................................125

Appendix 4. Nucleotide and protein sequences of the RMP1 (Os01g0533400)

gene............................................................................................................................129

Appendix 5. Nucleotide and protein sequences of the RMP2 (Os01g0899100)

gene............................................................................................................................131

Appendix 6. Nucleotide and protein sequences of the RMP3 (Os03g0381000)

gene ............................................................................................................................133

Appendix 7. Nucleotide and protein sequences of the RMP4 (Os04g0561900)

gene ............................................................................................................................135

Appendix 8. Nucleotide and protein sequences of the RMP5 (Os04g0650200)

gene ............................................................................................................................137

Appendix 9. Nucleotide and protein sequences of the RMP6 (Os06g0681100)

gene ............................................................................................................................139

Appendix 10. Nucleotide and protein sequences of the RMP7 (Os07g0664600)

gene ............................................................................................................................141

Appendix 11. Nucleotide and protein sequences of the RMP8 (Os12g0605900)

gene ............................................................................................................................144

Appendix 12. Nucleotide and protein sequences of the RMP9 (Os12g0637100)

gene ............................................................................................................................147

Appendix 13. Nucleotide and protein sequences of the RMP10 (Os12g0637900)

gene ............................................................................................................................149

VI

ACKNOWLEDGEMENT

First and foremost I would like to express my sincere gratitude to Prof.

Soon-Ki Park and Dr. Sung-Aeong Oh for all the guidance, helpful advice during

the whole period.

I would like to give a big thank to Prof. Ki-Hong Jung in Kuyng Hee

University for kindly provided microarray data profile, suggestions for my thesis.

I also would like to thank Prof. Jong-Tae Song, chairman of my advisory

committee, Prof. Jeong-Dong Lee, and Prof. Dong-Soo Park for their valuable

suggestions and critical review of my thesis.

In addition, I would like to thank all members in Sexual Plant Reproduction

Laboratory for their help.

Last, but not least, I wish to thank my wife, son, and family for their

support, understanding and encouragement during all this time.

Nguyen Tien Dung

VII

Identification and characterizationof microspore-preferred promoters in rice (Oryza sativaL.)

Nguyen, Tien-Dung

School of Applied Biosciences

The Graduate School, Kyungpook National University

Daegu, Korea

(Supervised by Professor Lee, Jeong-Dong)

(Abstract)

Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or

organ; however, their range of applications in other plant species has not been determined, to date. In this

study, I identified ten rice microspore-preferred (RMP1 to RMP10) promoters via meta-anatomical

expression analysis. I then investigated the expression of those promoters in transgenic rice (a homologous

system) and Arabidopsis (a heterologous system) using GFP-GUS reporter genes. As expected from

microarray data analysis, all of the ten RMP promoters directed similar GUS expression pattern in anthers,

GUS signals were detected from the microspore stage throughout the all stages of pollen development.

However, while four promoters, RMP2, RMP7, RMP9 and RMP10 did not direct GUS expressed in

vegetative tissues such as leaf, stem, root at seedling stage, the other six promoters conferred GUS activity in

those of seedlings. These results suggest that RMP promoters could be expressed preferentially in microspore

in rice. In contrast, RMP promoters directed GUS gene showing distinct expression patterns in Arabidopsis.

In inflorescence, the RMP2, RMP3 and RMP8 promoters directed GUS expression in young buds but not in

mature flowers. GUS signals were observed only at uni-cellular and bi-cellular stages of pollen development.

On the other hand, 2 promoters, RMP9 and RMP10, exhibited GUS expression in mature flowers at late￾pollen stages, tri-cellular and mature pollen. Whereas, the other five promoters, including RMP1, RMP4,

RMP5, RMP6, and RMP7 conferred GUS expression at all stages of pollen development, from uni-cellular

throughout mature pollen. The activity of these promoters was further examined in T2 seedlings. As a result,

seven promoters, except for RMP1, RMP2 and RMP10, showed GUS signals in shoot apical region or root

tissues of seedlings. In addition, analyzing promoter sequence revealed that the six most abundant CREs

detected in RMP promoter regions such as ACGTATERD1, ARR1AT, CAATBOX1, GATABOX,

MYBCORE, and DOFCOREZM. Moreover, eleven CREs related to organs/tissues preferred expression. Of

them, anther or pollen specific CREs such as GTGANTG10, POLLEN1LELAT52, SITEIIATCYTC,

5659BOXLELAT5659 were identified.

Furthermore, to verify the activity of promoters in microspore I carried out a functional

demonstration by performing a complementation analysis using a sidecar pollen (scp) mutant that displays

developmental defects at the microspore stage. Five promoters including the RMP1, RMP2, RMP3, RMP6

and OsLPS10 (rice late pollen specific promoter), which showed microspore expression in Arabidopsis, were

also verified. I found evidence that the OsLPS10, RMP1, RMP2, RMP3 and RMP6 promoters, which can be

VIII

an applied promoter in Arabidopsis, are useful for directing gene expression in the early stages of pollen

development. The results indicate that those promoters can direct the expression of target genes during the

stages of pollen development in rice, including early and late stages.

1

CHAPTER 1. GENERAL INTRODUCTION

Sexual reproduction is the most important characteristic of higher plants. It

is a process of producing offspring through fusion of male and female gametes in

flowers.

Floral structure

Flower is a critical reproductive organ of flowering plants. The structure of

rice flower consists of two main organs, male and female (Figure 1). Carpel or

pistil is the female reproductive organ which contains the stigma, a style and an

ovary. The stigma is a surface receptive to pollen grains connecting with the

ovary through a tubular structure called the style. The ovary is at the base of the

flower containing one ovule that becomes the seed after fertilization.

The male reproductive organs consist of six stamens. Each stamen has two

distinct parts; one is the filament and other is a pairs of anther that produces

pollen grains. At the mature stage, pollen grains is released from anthers and

transferred to the stigma. The anther is composed of highly specialized tissues for

pollen generation while the filament provides support for the anther. There are

two thecae in anther which are linked by connective tissue; each theca has two

locules that are connected by septum and stomium (Matsui et al. 1999).

Microsporogenesis and microgametogenesis are two sequential steps comprised

the male gametophytic development.

Before opening, the reproductive organs are protected by the lemma and

palea. Moreover, in rice, the lodicules are two wale-like, transparent, fleshy

structures located at the base of the flower adnate to the palea. They represent the

reduced calyx and corolla. At anthesis, the lodicules become turgid and thrust the

lemma and palea apart, allowing the elongating stamens to emerge above or

2

outside the open floret. The floral organs originate and attached to a placed on the

stem at the bottom part of the flower called rudimentary glumes (Figure 1).

3

Figure 1. Diagram of rice (Oryza sativa L.) floral structure.

4

Anther development

In rice, the development of anther is classified into eight stages (Ikeda et al.

2004; Itoh et al. 2005). Initially, the conformation of anther changes from ovoidal

into four-cornered shape and the archesporial cells (ACs) initiate at the

hypodermal layer of the anther. The ACs differentiates into primary sporogenous

cells (PSCs) and primary parietal cells (PPCs). After several rounds of division,

anther wall layer is formed. PPCs will continue to divide periclinally to generate

endothecium, a middle layer and a tapetum layer and will expand anticlinally.

After the anther wall is completed, the pollen mother cells (PMCs) undergo

meiosis to produce haploid gametes. Zhang and Wilson (2009) also classified the

rice anther development into eight stages based on the morphological cellular

landmark. However, with the results of light microscopy analysis of cellular

changes occurring in anther rice, the anther developmental courses was

categorized into 14 stages (Zhang et al. 2011, Zhang and Wilson 2009) similar to

the Arabidospsis anther development (Ma 2005, Sanders et al. 1999). At stage 1,

the floral meristem divides the cell in the L1, L2 and L3 layers, and then the

anther primordium is formed. The anther primordia continue cell division until

stage 5. The characteristic anther structure is formed and developed with locule,

wall, connective and vascular tissues in these stages. At stage 6, the secondary

sporogenous cells generate microspore mother cells (MMCs, also called PMCs)

within the locule. From stage 7 to 9, PMCs undergoes meiosis and forms dyads

and tetrads of haploid microspore. From stage 9, free microspore is released from

the tetrads with the degradation of callose wall. Early in this stage, microspores

are spherical with thin exines (Li and Zhang 2010). The microspore vacuolates

with an increase in volume forming round-shape at stage 10, and undergoes the

first mitotic division with asymmetric cell division at stage 11. Subsequently, the

generative cells separate from the pollen wall and move to the vegetative nucleus.

At the end of stage 11, the tapetum cells almost completely degenerate into

5

cellular debris and ubisch bodies on the internal surface. At stage 12, the

generative cell in the microspore divided into two sperm cells and mature pollen

formed with three nuclei. At stage 13, the lemma was opened and the anther

dehiscence occurred. At stage 14, the anther continued the release of mature

pollen grains. The genetic mechanism of stamen development is largely

conserved between rice and model eudicots (Yoshida and Nagato 2011). The

ABC model was first established to explain the genetic mechanism of floral

specification in Arabidospsis by Haughn and Somerville (1988). The sepals are

solely characterized by the expression of A gene, while two classes of genes (A

and B) worked together for the petals. The B and C genes are co-expressed for

stamen specification. Only C gene acts for the carpels. The ABC model was

further extended to the ABCDE or ABCE with E gene function encoded by

sepallata (SEP) and AGL16-like genes for development of all floral organs

(Yoshida and Nagato 2011). Most of the ABCDE genes belong to MADS box

family and are involved in floral development and evolution in higher plants

(Causier et al. 2010).

Male gametophyte development in higher plants

In higher plants, male gametophyte development is a complex process in

which gametophytic and sporophytic tissues are required. There are two distinct

phases of the male gametophytic life cycle, microsporogenesis and

microgametogenesis (Borg et al. 2009, Honys et al. 2006). The micro￾sporogenesis begins in the young anther where tetrads of four haploid

microspores are produced after dividing diploid pollen mother cells. Single

unicellular microspores are released from the tetrad by the activity of an enzyme

complex secreted by the tapetum at the end of this phase. During

microgametogenesis, the microspores grow and form a single vacuole. The role

of vacuole in microspore expansion is associated with polarization of the